Construction And Verification Of Fluorescent Tracer System Based On E-cadherin Gene Promoter

Epithelial mesenchymal transformation(EMT)refers to the process in which epithelial cells lose the tight connection between cells and the cytoskeleton undergoes rearrangement,thereby transforming into interstitial cells.As a procedural and reversible biological process,EMT appears in the process of embryonic development and organ formation,tissue repair and regeneration,and tumor cell invasion and migration.The occurrence of EMT is a dynamic process that interacts with transcription factors,mi RNAs,cell microenvironments,etc.to form a complex regulatory network.E-cadherin protein is encoded by the gene CDH1,its main role is to mediate cellcell adhesion,and is one of the main markers of epithelial cells.Studies have shown that E-cadherin is a tumor suppressor gene.Mutation and methylation of its promoter region all affect the expression of E-cadherin,which in turn affects the EMT process of tumors.Among them,TGF-β plays an important role in regulating cell activity and is a classic inducing factor that induces EMT in tumor cells.Therefore,this paper mainly uses E-cadherin as a marker and uses the inducible factor TGF-β1 to verify the EMT tracer model.In this study,a fluorescent tracing system with the E-cadherin promoter was mainly constructed,and the feasibility of the tracing system was proved,and the system was used to confirm that the polypeptide M1-138 can inhibit EMT.First,it was verified that A549 cells and MCF-7 cells can effectively respond to the induction of the classic EMT-inducing factor TGF-β1,and these two cells were selected as model cells;then,the lentiviral vector of the E-cadherin promoter was constructed and packaged with Plasmids p VSVG and Δ8.91 were co-transfected into HEK293 T cells,packaged with lentivirus,and formed lentiviral particles with infectious ability,which further infected A549 and MCF-7 cells to construct stable cell lines.Then.After 48 hours of induction of the two cell lines with TGF-β1,it was found that the intensity of green fluorescence decreased and the expression of E-cadherin and GFP at the m RNA and protein levels decreased.Therefore,the tracer system of E-cadherin promoter was effective and feasible.Finally,the purified polypeptide M1-138 was added to the two cell lines,and the green fluorescence intensity of the two cells was increased,and the expression of E-cadherin and GFP at the m RNA and protein levels increased,indicating that M1-138 can promote the expression of E-cadherin.,Effectively inhibit the cell’s EMT process.In summary,the E-cadherin promoter is used to trace EMT.This process is simple and fast.It can effectively screen out small molecule drugs and peptides related to EMT,which lays the foundation for the study of EMT process of tumor.